Alexa fluor 488 conjugated anti mouse igg a 11001

For confocal microscopy, cells were fixed in 4% paraformaldehyde and incubated in blocking buffer containing 5% donkey serum and 1% bovine serum albumin. After incubation with rabbit anti-RIPK1 or anti-RIPK3 and mouse anti-LMP1 antibodies, Alexa Fluor 594-conjugated anti-rabbit IgG (A-21207) and Alexa Fluor 488-conjugated anti-mouse IgG (A-11001) purchased from Thermo Fisher were added, respectively. The cells were incubated with DAPI from Sigma-Aldrich to stain nuclei. Images were acquired on a Leica TCS SP5 confocal microscope. For TEM, cells were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2). The cells were washed in 0.1 M cacodylate buffer, then fixed with the same buffer with 1% osmium tetroxide, washed, and dehydrated through a graded series of ethanol to 100% and embedded in epoxy resin. The sections were counterstained with uranyl acetate and lead citrate. Images were acquired on a Hitachi HT7700 TEM operated at an accelerating voltage of 100 kV.

Immunostaining was performed using anti-FTL (MA5-14733; Thermo Fisher Scientific) and anti-GFP monoclonal antibodies (D153-3; MBL) as primary antibodies, and Alexa Fluor 488-conjugated anti-mouse IgG (A-11001; Thermo Fisher Scientific), Alexa Fluor 488-conjugated anti-rat IgG (A-11006; Thermo Fisher Scientific), and Alexa Fluor 568-conjugated anti-mouse IgG (A-11004; Thermo Fisher Scientific) antibodies as secondary antibodies. HeLa cells grown on a cover glass were washed twice with phosphate-buffered saline (PBS), treated with 4% paraformaldehyde (09154-85; Nacalai Tesque) for 10 min at room temperature, washed three times with PBS, treated with 50 μg/ml digitonin for 5 min at room temperature, washed three times with PBS, and treated with 3% BSA in PBS for 30 min at room temperature. The cells were incubated with primary antibodies in PBS containing 3% BSA for 1 h at room temperature, washed five times with PBS, incubated with second antibodies in PBS containing 3% BSA for 1 h at room temperature, washed five times with PBS, and then treated with SlowFade Gold Antifade Mountant with DAPI (S36939; Thermo Fisher Scientific).